CD34 is expressed specifically on human hematopoietic stem cells and not on mature blood cells. CD34+ cells can reconstitute normal hematopoiesis of all cell lines, and to date it represents the best defined human stem cell marker; therefore, it is a model for stem cell gene expression. Previous studies have defined the some of the promoter elements for human and murine CD34, but the regulation of this and other stem cell genes are still very poorly understood. The goal of this project is to continue to define and analyze the control elements of CD34, and to characterize those elements of the CD34 gene which can direct stem cell expression of heterologous genes. Therefore, the specific aims are to characterize and study the regulatory elements of CD34 in the following steps: (1) To perform a comparative analysis of the human and murine CD34 genes, comparing DNA sequence, DNAse I hypersensitive sites, and performing cross hybridization studies; (2) To identify the important control elements regulating CD34 gene expression in stem cells, testing them in transgenic mice with human CD34 PAC clones, characterizing CD34 control elements which direct expression of a reporter gene in stem cells, and characterizing DNA binding proteins which regulate these elements; and (3) To use homologous recombination to insert heterologous genes into CD34 instructs for delivery into the stem cell compartment. The characterization of these elements could lead to the development of new vectors for gene therapy, as well as new understanding of the factors regulating genes in stem cells.